Investing Early Signaling Events in IgE-FcɛRI Activation Using SEM

Abstract

Antigen-mediated cross-linking of immunoglobulin E (IgE) bound to its high affinity receptor FcɛRI on mast cells initiates a transmembrane signaling cascade that results in cell activation and exocytotic release of chemical mediators involved in allergic response. Plasma membrane lipids and proteins redistribute as part of this transmembrane signaling process. To understand the functional role of these redistributions, resolution of their size, composition and structure on the nanometer scale is required. We utilize high resolution scanning electron microscopy (SEM) to directly visualize sub-micron membrane domains in intact cell membranes. In our experiments, the distribution of gold-labeled proteins and lipids is analyzed at the surface of intact fixed cells using backscattered electron detection. In parallel, we also observe membrane topography using secondary electron detection. We use a pair-correlation function analysis to quantify protein distributions and parameterized domain size. We have mapped the distribution of a variety of proteins, both related and non-related to the IgE signaling pathway. Using this experimental and quantitative method, we observe dramatic changes in the nano-scale membrane distribution of IgE due to stimulation with multivalent ligands. In resting cells, IgE receptors are clustered into small domains less than 30nm. After stimulation, receptors redistribute into large domains that are correlated at long length-scales and subsequently reduce in size at long stimulation times. We also observe cross-linking-dependent rearrangement of several inner leaflet-associated proteins implicated in early signaling events. In contrast, outer leaflet GPI-anchored proteins are not affected. We have also quantified the co-redistribution of IgE with other membrane proteins after stimulation using cross-correlation functions. These findings provide valuable insights into the mechanisms that drive the selective nanoscopic reorganization of plasma membrane proteins during immune cell signaling.